Biohythane production via anaerobic digestion process: fundamentals, scale-up challenges, and techno-economic and environmental aspects

Biohythane, a balanced mixture comprising bioH2 (biohydrogen) and bioCH4 (biomethane) produced through anaerobic digestion, is gaining recognition as a promising energy source for the future. This article provides a comprehensive overview of biohythane production, covering production mechanisms, microbial diversity, and process parameters. It also explores different feedstock options, bioreactor designs, and scalability challenges, along with techno-economic and environmental assessments. Additionally, the article discusses the integration of biohythane into waste management systems and examines future prospects for enhancing production efficiency and applicability. This review serves as a valuable resource for researchers, engineers, and policymakers interested in advancing biohythane production as a sustainable and renewable energy solution.


Introduction
The rapid increase in energy consumption is depleting natural resources and causing a significant rise in greenhouse gas emissions.To tackle the challenges posed by fossil fuels, the world is shifting towards a more sustainable and cleaner energy system.Recent statistics show that global renewable energy capacity reached 3,869,705 megawatts (MW) in 2023, marking a 128% increase from 2014 (IRENA 2024).This growth is attributed to various renewable energy sources, as detailed in Table 1.Furthermore, global renewable energy consumption has surged, with levels nearing 45.18 exajoules in 2022, a notable 227% increase from 2012 (Energy Institute 2023).
To develop a more sustainable energy system, the use of hydrogen (H 2 ) is being explored extensively.H 2 is considered a highly efficient and clean energy carrier with a calorific value of 142 MJ/kg and only water as a combustion by-product (Silva et al. 2017).Methane (CH 4 ) is also being investigated as a clean and safe alternative to petroleum and diesel, widely utilized in various industries such as chemical and transport sectors (Abdur Rawoof et al. 2021).Unlike traditional fuels, the combustion of H 2 and CH 4 does not result in the emission of major air pollutants like nitrous oxides (NO X ) and sulfur dioxide (SO 2 ) (Krishnan et al. 2019;Mozhiarasi et al. 2023).However, CH 4 has limitations such as slow burning speed, narrow flammability range, and high ignition temperature, leading to lower combustion efficiency and higher energy consumption in CNG-powered vehicles (Bauer and Forest 2001).By adding even a small amount of H 2 to CH 4 , the flammability range is extended, and ignition time is reduced due to H2's higher mass-specific heating value and faster flame speed compared to CH 4 .Additionally, mixing H 2 with CH 4 increases the hydrogen-to-carbon ratio, resulting in decreased greenhouse gas emissions (Hans and Kumar 2019).Furthermore, blending CH 4 with H 2 reduces the risks of abnormal combustion like flashback and engine explosion, common issues in H 2 combustion (Makaryan et al. 2022).
Hythane, a blend of H 2 and CH 4 , has emerged as a promising energy carrier for the transition to a Responsible Editor: Ta Yeong Wu hydrogen-based economy.This valuable energy carrier was initially developed by Hydrogen Component Inc. (HCI) in the early 1990s as a fuel for powering internal combustion engines (Bolzonella et al. 2018).In 2004, the patent and trademark for hythane were acquired by Eden Energy, leading to the establishment of Hythane™ Company LLC (now Eden Innovations LLC).Following this acquisition, several pilot-scale projects were launched globally (McWilliams 2017).The Montreal hythane bus project successfully reduced NO x emissions by utilizing hythane with a 10% (v/v) H 2 content.Compared to CNG-powered buses, the use of hythane resulted in a 45% decrease in NO x emissions.Similarly, the Sun Line transit agency project in California demonstrated the environmental benefits of hythane as an alternative fuel.By using hythane with over 20% (v/v) H 2 content, the project achieved a significant 50% reduction in NO x emissions compared to CNG-powered buses (Abdur Rawoof et al. 2021).The Fiat Company also developed a car with a 900 cc two-cylinder engine using hythane with 30% H 2 (v/v), achieving low CO 2 emissions of only 69 g/ km 2 (Bolzonella et al. 2018).Some of the proven advantages of hythane in the automotive sector include lower energy requirements for engine combustion and increased heat and fuel efficiency (Abdur Rawoof et al. 2021).Additionally, the use of hythane as a vehicle fuel does not necessitate any special storage system or significant modifications to existing CNG engines and infrastructure (Krishnan et al. 2019).The superior properties of hythane have led to its commercialization as a transport fuel in various countries, including the USA and India.Furthermore, its potential as an alternative fuel source has attracted the interest of major automobile manufacturers, such as Fiat, Volvo, and Toyota (Shanmugam et al. 2021a).Table 2 presents a summary of the advantages and disadvantages associated with hythane.
Currently, hythane production is mainly done using energy-intensive thermochemical processes that rely on fossil fuels like natural gas (Shanmugam et al. 2021a).However, there is a shift towards more sustainable methods, including biological production, to address the drawbacks of -Increased flame speed and reduced combustion duration -Overcoming high ignition temperatures -Higher thermal efficiency compared to natural gas -Greater braking efficiency and power output over natural gas -Minimization of cycle-by-cycle variations in engines -Reduced fuel consumption -Reducing emissions of unburned hydrocarbons, CO 2 , and CO -Possible utilization without significant modifications to existing natural gas infrastructure and CNG engines -Potential for increased NO x emissions at higher hydrogen contents without adequate tuning -Dependence of total CO 2 emissions on the method of hydrogen production -Increased heat transfer to cylinder walls and a decrease in engine power -Increased ignition delay time at higher hydrogen contents, necessitating careful engine calibration -Need for careful handling and storage due to hydrogen's flammability and diffusivity -Requirement for modifying existing storage and supply infrastructure to handle higher hydrogen contents Akansu et al. (2004); Genovese et al. (2011); Villante and Genovese (2012); Biffiger and Soltic (2015); Yadav and Sircar (2017); Makaryan et al. (2022) these processes.The biological production of hythane from renewable resources like biomass through anaerobic digestion (AD) offers significant sustainability advantages over fossil fuel-based processes (Alavi-Borazjani et al. 2023).This process can produce both H 2 and CH 4 , which can be used independently or combined to create biohythane, a biologically derived hythane mixture (Liu et al. 2013).The potential benefits of biological hythane production have spurred active research in the field.As shown in Fig. 1, from 2010 to 2022, there was a consistently increasing trend in biohythane publications, rising from only one in 2010 to 92 in 2022.However, in 2023, there was a slight decrease, with 70 articles published according to data from the Science Direct database.Despite this slight dip, the overall trend indicates a growing interest in biohythane research, emphasizing the need for continued investment and exploration in this area.This comprehensive review pushes the boundaries of biohythane production research by moving beyond the existing body of knowledge.While acknowledging the important groundwork laid by previous reviews, it dives deeper into the topic, carefully selecting distinct and crucial information from a wider array of recent studies.By presenting data and insights not previously covered, this work offers a fresh perspective on the multifaceted domain of biohythane production via the AD process.This meticulous approach aims to provide researchers with a thorough and up-to-date one-stop resource, streamlining the process of literature searches and cross-referencing.As a result, this review serves as a valuable tool, equipping researchers with a well-rounded perspective on the current landscape of biohythane generation through AD.

Mechanisms of biohythane production
Anaerobic digestion (AD) involves the biological breakdown of organic matter by microorganisms in an oxygen-free environment, resulting in the production of biogas.The process typically consists of hydrolysis followed by acidogenesis, acetogenesis, and finally methanogenesis (Tsigkou et al. 2022).Figure 2 illustrates the key steps of the AD process.
During hydrolysis, complex organic compounds are enzymatically broken down into simpler constituents, including monosaccharides, amino acids, and fatty acids (Alavi-Borazjani et al. 2021a, 2021b).In the acidogenesis phase, the products of hydrolysis are transformed by acidogenic bacteria into a mixture of organic intermediates like volatile fatty acids (VFAs), lactic acid, and alcohols, as well as inorganic intermediates such as H 2 , CO 2 , hydrogen sulfide (H 2 S), and ammonia (NH 3 ) (Madsen et al. 2011).In the acetogenesis phase, acetogenic bacteria further process the products from the previous step, leading to the production of acetic acid (CH 3 COOH), H 2 , and CO 2 .In the concluding phase, referred to as methanogenesis, diverse microorganisms consume acetate, CO 2 , and H 2 to generate CH 4 (Jain et al. 2015).
The synthesis of biohythane using the AD process can be achieved through either a single-stage or a two-stage approach, as shown in Fig. 3.

Single-stage AD
In the single-stage AD process, the four stages of organic material conversion are integrated into a unified system (Nagao et al. 2012).The idea of co-producing bioH 2 and bioCH 4 in a single reactor to create sustainable hythane was proposed in 2015 (Ta et al. 2020).Single-stage AD offers advantages such as simplicity in design and reduced operating costs (Kabir et al. 2022).Research indicates that single-stage AD has led to interesting dynamics in microbial functions and interactions for biohythane production (Lay et al. 2020).However, a significant drawback is the risk of reactor acidification from excessive VFA formation during acidogenesis and acetogenesis.This can occur when VFA concentrations surpass a certain threshold, causing pH reduction, toxicity to hydrogen producers, and decreased H 2 yield (Hans and Kumar 2019).To overcome the limitations of single-stage systems in biohythane production, a new two-compartment bioreactor has been developed (Vo et al. 2019).The upper and lower compartments serve specific roles in the production process, with the upper compartment dedicated to bioCH 4 generation and the lower compartment focused on bioH 2 refinement.This improved design allows for better control, enhanced reaction kinetics, and ultimately, superior biohythane production by segregating the two stages in separate compartments.Another promising approach in single-stage biohythane production involves entrapping hydrogenic and methanogenic bacteria separately (Ta et al. 2020;Nguyen et al. 2022).This concept entails trapping concentrated populations of hydrogenic and methanogenic bacteria within their respective beads.The use of the cell entrapment technique provides a protective environment for microbes while allowing substrate and metabolic product diffusion (Park and Chang 2000).By employing this method, the challenges associated with hydrogenotrophic methanogenesis can be mitigated, as the physical separation of hydrogenic and methanogenic bacteria enables better control and optimization of their metabolic activities (Ta et al. 2020).

Two-stage AD
The two-stage AD concept was originally introduced by Travis in 1904 for wastewater treatment and has since been adapted by industries for both pre-treatment and post-treatment purposes (Rajendran et al. 2020).Recently, there has been a growing interest in applying this approach to biohythane production.In this process, the initial stageshydrolysis, acidogenesis, and acetogenesis-are combined in a single reactor to produce bioH 2 .Subsequently, methanogenesis, the final step, occurs in a separate reactor using the spent medium from the first stage to generate bioCH 4 (Shanmugam et al. 2021a).
A dual-stage system for biohythane production offers several advantages over a single-stage system.Firstly, separating bioH 2 and bioCH 4 production into two distinct stages enhances process stability (Chu et al. 2010;Sasidhar et al. 2022).Each stage can be controlled independently, optimizing environmental conditions and microbial activities.This separation reduces potential inhibitory effects between acidogenic and methanogenic bacteria, minimizing process disruptions and improving stability.Secondly, the two-stage process shortens fermentation time and allows for a higher organic loading rate, increasing biohythane production efficiency (Ueno et al. 2007b;Rajendran et al. 2020).In the initial stage, acidogenic bacteria rapidly convert organic matter into VFAs, accelerating the breakdown of complex organic compounds.This shorter first stage enables earlier commencement of the methanogenesis stage, leading to a quicker overall fermentation period.Additionally, the rapid conversion of organic materials in the first stage allows for a higher feedstock input, facilitating an increased organic loading rate for improved productivity.Thirdly, by leveraging the unique advantages of each stage and optimizing the twostage AD system, energy recovery from organic matter can be significantly enhanced (Kvesitadze et al. 2012;Hans and Kumar 2019).These benefits make the twostage system a more efficient and sustainable approach to biohythane production.Table 3 summarizes the advantages and disadvantages of both single-stage and twostage AD.

BioH 2 and bioCH 4 production pathways
The initial stages of AD, including hydrolysis, acidogenesis, and acetogenesis, exhibit similarities to dark fermentation (DF) for generating bioH 2 gas.In the DF process utilizing glucose as the substrate, hydrogen-producing bacteria metabolize glucose into pyruvate through glycolytic pathways.During the conversion process, the system produces adenosine triphosphate (ATP), the main energy carrier in cells, from adenosine diphosphate (ADP), a precursor molecule for energy production, and nicotinamide adenine dinucleotide (NADH) in its reduced form, which is essential for electron transport (Ma et al. 2015;Srivastava et al. 2021).Equation 1 illustrates the specific details of this biochemical pathway.Obligate anaerobes then convert pyruvate into acetyl coenzyme A (acetyl-CoA) using the enzyme pyruvate-ferredoxin oxidoreductase (PFOR) and ferredoxin (Fd), leading to the production of H 2 by hydrogenase (Eqs.( 2) and ( 3)) (Ma et al. 2015).In contrast, facultative anaerobes   4) and ( 5) (Ntaikou et al. 2010;Liu et al. 2017).
The quantity of H 2 produced depends on the final product of pyruvate oxidation.When acetate is the exclusive end product, four moles of H 2 are generated per mole of glucose.In contrast, if the end product is butyrate, only two moles of H 2 are formed (Eqs.( 6) and ( 7)) (Alavi-Borazjani et al.

2019; Chozhavendhan et al. 2020).
There are three primary pathways for methanogenesis: (1) hydrogenotrophic, (2) acetoclastic, and (3) methylotrophic (Chen et al. 2022).In hydrogenotrophic methanogenesis, CO 2 is reduced to CH 4 using H 2 or formate as electron donors (Ferry 2011).The reduced ferredoxin plays a crucial role in the initial step of methanogenesis, where CO 2 is reduced to a formyl group attached to the methanofuran (MFR) carrier molecule.The formyl group is subsequently moved to the tetrahydromethanopterin (H 4 MPT) carrier.This transfer induces cyclization through dehydration, resulting in the formation of methenyl-H 4 MPT.The methenyl group undergoes a two-step reduction process, initially forming a methylene and subsequently a methyl group.The resulting methyl group is then attached to coenzyme M (HS-CoM), which contains a sulfhydryl group.Finally, coenzyme B (HS-CoB) oxidizes HS-CoM, leading to the formation of CH 4 and a heterodisulfide intermediate (CoM-S-S-CoB).This heterodisulfide undergoes further reduction to restore HS-CoM and HS-CoB (Costa and Leigh 2014).In hydrogenotrophic methanogenesis, a key methyl transfer reaction within the core pathway is directly linked to energy capture.The coenzyme M methyltransferase (Mtr) translocates sodium ions across the membrane, creating a sodium driving force that is utilized by an ATP synthase (Kurth et al. 2020).In acetoclastic methanogenesis, acetate is transported into microbial cells and converted to acetyl-CoA through acetate kinase/phosphotransacetylase or acetyl-CoA synthetase (Berger et al. 2012).The acetyl-CoA decarbonylase/synthase complex then cleaves the acetyl group in a dismutation reaction.This reaction generates CO 2 and channels the remaining methyl group toward the central methanogenic pathway for conversion to CH 4 (Kurth et al. 2020).Energy conservation in this process involves membrane-bound methyltransferase Mtr and a membranebound electron transport chain utilizing reduced ferredoxin and heterodisulfide (Welte and Deppenmeier 2014).Acetoclastic methanogenesis transports more sodium/proton ions per cycle compared to hydrogenotrophic methanogenesis but requires an initial ATP investment during acetate activation.In methylotrophic methanogenesis, monocarbon compounds like methanol, methylamines, and methyl sulfides function as both electron donors and acceptors (Söllinger and Urich 2019).The pathway begins with these substrates entering as methyl-S-CoM.The reduction of methyl-S-CoM to CH 4 involves transferring electrons either from H 2 or through oxidizing another molecule of methyl-S-CoM to CO 2 in a process known as methyl disproportionation (Costa and Leigh 2014).Three-quarters of the methyl groups are reduced to CH 4 in methylotrophic methanogenesis, with the remaining quarter oxidized to CO 2 .Energy conservation occurs through membranebound electron transport, with membrane-bound methyltransferase functioning in the reverse reaction, dissipating the proton/sodium driving force (Kurth et al. 2020).A common step across all methanogenesis pathways is the reduction of methyl-S-CoM and the breakdown of CoM-S-S-CoB.Figure 4 provides a simplified illustration of the three methane production pathways.

Microbial diversity in biohythane production
The generation of bioH 2 and bioCH 4 through the AD process is influenced by an intricate interplay of various microorganisms, each having distinct environmental needs.To attain the targeted biohythane makeup with the ideal bioH 2 /bioCH 4 ratio, precise management of the growth of these microorganisms according to their individual traits is essential.More information regarding the particular microorganisms participating in both hydrogenogenic and methanogenic processes is outlined below.

Microorganisms in bioH 2 production process
Various microorganisms play a role in bioH 2 production, including strict anaerobes, facultative anaerobes, and even

Acetyl-CoM Acetate
certain aerobes (Ghimire et al. 2015).Clostridia are the primary bioH 2 -producing microorganisms under mesophilic conditions (Fang et al. 2002).These spore-forming obligate anaerobes have a shorter doubling time and greater resilience under unfavorable conditions compared to other anaerobic bacteria, making them ideal for industrial applications (Roy and Das 2016).Additionally, Clostridia can degrade crystalline cellulose, a highly resistant substrate (Wei et al. 2014).Enterobacter, a non-sporulating facultative anaerobe with a faster growth rate than obligate anaerobes, is another significant bioH 2 producer (Hans and Kumar 2019).Enterobacter bacteria can utilize various carbon sources as substrates, but there are metabolic differences between them and Clostridium sp., especially in the fermentation by-products (Tapia-Venegas et al. 2015).Studies have shown that Enterobacter sp. has a lower bioH 2 production yield compared to Clostridium sp.(Abdur Rawoof et al. 2021) and is more sensitive to traces of dissolved oxygen (Roy and Das 2016).Within the Bacillus genus, which are typically facultative anaerobes, robust H 2 -producing bacteria like Bacillus licheniformis and Bacillus coagulans have been identified.Their ability to thrive in the presence of dissolved oxygen makes them attractive for industrial applications compared to strict anaerobes.E. coli has been widely used for genetic manipulation to enhance bioH 2 production.For example, overexpression of formate hydrogen lyase (FHL) in E. coli increased bioH 2 formation by 2.5-fold (Yoshida et al. 2005).Similarly, modifying the metabolic pathways of E. coli by selectively removing certain genes resulted in a fivefold enhancement in bioH 2 production (Tran et al. 2014).In thermophilic dark fermentation, Thermoanaerobacter sp., Thermoanaerobacterium sp., and Clostridium sp. are the dominant bacterial species responsible for bioH 2 production (Mozhiarasi et al. 2023).Caldicellulosiruptor sp. and Thermotoga sp. are examples of obligate fermentative anaerobes known for their bioH 2 production under extreme thermophilic conditions (Brynjarsdottir et al. 2013).
In dark fermentation systems, various microorganisms play a role in enhancing bioH 2 production, even though they do not directly produce bioH 2 .For instance, Streptococcus sp. has been reported to enhance bioH 2 production through granule formation (Hung et al. 2011a).Similarly, Bifidobacterium sp.aids in biohydrogenation by decomposing complex organic compounds into smaller molecules suitable for uptake by hydrogen-producing bacteria (Cheng et al. 2008a;Doi et al. 2009).Studies have also shown that the presence of Klebsiella sp. in the fermentation medium can create anaerobic conditions by removing oxygen, which is advantageous for obligate bioH 2 -producing bacteria like Clostridium sp.(Hung et al. 2011b).Table 4 provides a list of significant microorganisms involved in bioH 2 production, including both direct H 2 producers and those that facilitate hydrogen generation, along with their key characteristics.
It is important to understand that the roles of coexisting microorganisms in bioH 2 production can be intricate and dependent on the context.For instance, Bacillus sp. has been found to have both positive and negative impacts on bioH 2 production in various fermentation systems (Hung et al. 2011a).Therefore, the influence of coexisting microorganisms on bioH 2 formation is not always straightforward and requires careful consideration.Additionally, when dealing with mixed cultures, there is a possibility of undesired microorganisms coexisting with bioH 2 producers, which can hinder bioH 2 formation or consume bioH 2 (Tapia-Venegas et al. 2015).However, this issue can be addressed by treating the inoculum before use.Figure 5 depicts different pre-treatment techniques for enhancing the population of bioH 2 -producing bacteria.Among these methods, heat shock treatment is the most commonly utilized.This method involves subjecting the sample to high temperatures for a specific duration, followed by a gradual cooling process to return it to room temperature.Previous studies have shown that optimal temperature and duration for pre-treatment can vary depending on the inoculum source.Reported temperatures range from 65 °C (Baghchehsaraee et al. 2011) to 121 °C (Wang et al. 2003), with treatment durations as short as 10 min (Cavalcante de Amorim et al. 2009) and as long as 5 h (Argun et al. 2008).Another common pre-treatment strategy for dark fermentative hydrogen production utilizes acid or base shocks to selectively eliminate unwanted microorganisms, especially methanogens, from the seed sludge.According to existing literature, acid-shock treatment typically involves a pH range of 2 (De Sá et al. 2013) to 4 (Sen and Suttar 2012) and exposure duration of 30 min (Zhu and Béland 2006) to 24 h (Elbeshbishy et al. 2010).Similarly, base-shock treatment typically utilized pH levels between 10 and 12 and exposure times ranging from 30 min (Zhu and Béland 2006) to 24 h (Kan 2013).The effectiveness of acid/base-shock treatment for enriching hydrogen producers remains a topic of debate, as existing literature does not provide consistent conclusions.While some studies have shown better results with acid-shock treatment (Chang et al. 2011), others have found base-shock treatment to be more effective (Zhu and Béland 2006;Wang and Wan 2008a;Yin et al. 2014).Moreover, while some studies have indicated that pH treatment can lead to higher bioH 2 yield compared to heat treatment, the general consensus is that heat treatment is a more effective method for enhancing the bioH 2 production capacity of the inoculum (Wang and Yin 2017).

Microorganisms in bioCH 4 production process
Archaea, specifically those within the Euryarchaeota phylum, are the primary microorganisms responsible for methanogenesis.These microorganisms have distinct characteristics that set them apart from bacteria and eukaryotes (Roy  (Angelidaki et al. 2011).
Most methanogens utilize H 2 as both an electron donor and energy source to convert CO 2 into CH 4 through hydrogenotrophic activity.It is interesting to note that approximately 45% of hydrogenotrophs have the ability to use formate as an alternative to H 2 (Megonigal et al. 2014).Some hydrogenotrophs from the order Methanomicrobiales, such as Methanoculleus sp. and Methanogenium sp., can function as mixotrophs using a combination of carbon and energy sources (Angelidaki et al. 2011).Acetate oxidation accounts for about two-thirds of the CH 4 produced in anaerobic systems (Pavlostathis 2011), with only two genera from the Methanosarcinales order, Methanosaeta, and Methanosarcina, involved in acetoclastic methanogenesis (Welte and Deppenmeier 2014).Methanosaeta is an obligate acetateconsuming methanogen that thrives in environments with low acetate concentrations, while Methanosarcina is versatile and capable of utilizing various carbon sources besides acetate (Kurade et al. 2019).Methylotrophic methanogenesis is primarily found in the Methanosarcinales order, with the exception of Methanosphaera, a genus from the Methanobacteriales order (Conrad 2020).
Most of the known methanogens are either mesophilic or moderately to extremely thermophilic, with only a few species able to survive and produce CH 4 at low temperatures (Simankova et al. 2003;Angelidaki et al. 2011).The order Methanobacteriales consists of two families, Methanobacteriaceae and Methanothermaceae.While members of the Methanobacteriaceae family do not thrive at temperatures above 70 °C, the Methanothermobacter genus within this family is prevalent in mildly warm environments and thrives best around 65 °C.In contrast, all members of the Methanothermaceae family have an optimal growth temperature range of 83 to 88 °C.The Methanococcales and Methanopyrales orders also include species that excel at temperatures exceeding 70 °C.For example, Methanocaldococcus and Methanotorris, two genera within the Methanococcales order, can flourish at temperatures ranging from 80 to 88 °C (Topçuoglu and Holden 2019).Methanopyrus kandleri, an exceptionally heat-tolerant strain from the Methanopyrales order, can even survive at 122 °C.On the other hand, Methanogenium frigidum from the Methanomicrobiales order is a psychrophilic strain that is highly cold-resistant, capable of thriving between 0 and 17 °C with an optimal temperature of 15 °C (Lyu et al. 2018).Building on the discussion of specific methanogens in this section, Table 5 summarizes their taxonomic classification (order) and key characteristics for easy reference.
pH pH is considered the most sensitive biochemical factor, influencing the enzymatic machinery of microorganisms and maintaining cell redox potential in AD (Yang et al. 2015;Vongvichiankul et al. 2017).A significant issue in the dark fermentation process is the continuous formation of volatile fatty acids (VFAs), resulting in a significant drop in pH levels and disruption of microbial membranes (Yeshanew et al. 2016;Khan et al. 2018;Kumar et al. 2021).The ideal pH for bioH 2 generation is typically within a range of 6.0 to 8.0 (Sinha and Pandey 2011), although some strains can produce bioH 2 even in acidic environments below pH 6.0 (Pandey et al. 2009).At pH levels below 4.5, a metabolic shift from acidogenesis to solventogenesis can occur, negatively impacting bioH 2 formation (Khanal et al. 2004;Van Ginkel and Logan 2005).BioH 2 production may cease completely at pH levels of 3.8-4.2(Shanmugam et al. 2021b;Ghosh and Kar 2022).
For bioCH 4 production, a pH range of 6.7-7.5 is recommended for optimal methanogen activity (Alavi-Borazjani et al. 2020;Jadhav et al. 2021), as methanogens struggle to thrive below pH 6.5 and above pH 9 (Kabir et al. 2022).Incomplete breakdown of organic matter during the acidogenesis stage disrupts the subsequent methanogenesis stage.This inefficiency can result in the build-up of VFAs and other organic acids, as the microbial populations responsible for converting these intermediates into methane are either not functioning optimally or are overwhelmed.The accumulation of these organic acids results in an increased concentration of hydrogen ions (H + ), leading to a reduction in pH.This acidic environment further impairs methanogenic activity, creating a vicious cycle.As acidification progresses, more acidic ions dissociate, accelerating the decline in pH.If left unchecked, this cycle can ultimately halt biological methane production (van Lier et al. 2020;Sasidhar et al. 2022).
Maintaining optimal pH levels in AD processes has traditionally relied on the use of chemicals like sodium hydroxide (NaOH), potassium hydroxide (KOH), and sodium carbonate (Na 2 CO 3 ) (Xie et al. 2014).However, a more cost-effective approach has emerged with the development of digestate recirculation systems.These systems strategically reintroduce digestate, a nutrient-rich by-product of the methanogenic stage, back into the acidogenic reactor (Aslanzadeh et al. 2013;Algapani et al. 2019;Liu et al. 2019).This recirculation also accelerates substrate degradation, allowing for higher organic loading rates and reducing the required reactor volume (Zuo et al. 2013).However, excessive recirculation can lead to ammonia build-up, which can inhibit both hydrogenogenic and methanogenic processes (Cavinato et al. 2012;Wu et al. 2018).Conversely, very low recirculation ratios may not provide enough buffering capacity to control pH levels effectively (Bolzonella et al. 2018).

Temperature
Temperature plays a crucial role in the success of two-stage AD for biohythane production.It significantly influences various aspects of the process, including the properties of microbial cells, the types of metabolic by-products generated, and the specific nutrient requirements of the microbes involved (Kumar et al. 2021).Enzymes have an optimal temperature range for their activity, and any deviation from this range can result in denaturation or enzyme inactivation, hindering the process and halting metabolic product production (Shanmugam et al. 2021b).Enzyme activity typically increases by up to twofold for every 1 °C temperature rise until reaching the optimum temperature.However, surpassing the optimal temperature leads to a decline in enzyme activity (Roy and Das 2016).Therefore, optimizing the operational temperature is crucial for improving bioH 2 and bioCH 4 production processes.
Microorganisms involved in bioH 2 production exhibit a remarkable range of optimal growth temperatures.Psychrophiles, for example, thrive in cold environments below 25 °C.Mesophiles function best in moderate temperatures ranging from 25 to 45 °C.Thermophiles, on the other hand, prefer warmer conditions between 45 and 65 °C.Finally, extreme thermophiles demonstrate optimal activity at high temperatures, between 65 and 80 °C.While hyperthermophiles exist and can tolerate temperatures exceeding 80 °C, their extreme temperature requirements make them less commonly employed in bioH 2 production processes (Alavi-Borazjani et al. 2021a, 2021b).Thermophilic conditions (typically around 55 °C) offer a significant advantage for bioH2 production.This stems from the temperature dependence of hydrogenase, the key enzyme responsible for bioH 2 generation.Hydrogenase exhibits its peak activity at these elevated temperatures, maximizing the efficiency of the bioH 2 production process (Reith et al. 2003).As previously stated, creating thermophilic conditions offers an additional benefit in bioH 2 production by selectively promoting the growth of bioH 2 producers while inhibiting the growth of competing microorganisms.This is due to the fact that most bioH 2 producers can form spores and survive in harsh conditions like high temperatures, whereas many unwanted hydrogen-consuming microorganisms are sensitive to high temperatures and can be deactivated (Liu et al. 2018).Increasing operational temperatures has been demonstrated to enhance the breakdown of stubborn organic  matter like lipids (Chipasa and Mȩdrzycka 2006).Additionally, elevated temperatures can lead to an increase in reactor pH, which can significantly enhance microbial activity in the bioH 2 production process (Ventura et al. 2014).
Thermodynamic principles can help explain the impact of temperature on bioH 2 production.Specifically, the standard enthalpy change (ΔH°) during the conversion of glucose to acetate can be examined, assuming a theoretical yield of 4 moles of H 2 per mole of glucose.This reaction is endothermic (ΔH° > 0), meaning it requires an input of energy to proceed.In this case, energy is absorbed in the form of heat, and the products have a higher enthalpy than the reactants (Sarangi and Nanda 2020).The Van't Hoff equation (Eq.( 8)) (Smith et al. 2018), which describes the relationship between temperature and the equilibrium constant (K) for a chemical reaction, can also be used to elucidate the thermodynamics of bioH 2 production: where K 1 and K 2 are the equilibrium constants at temperatures T 1 and T 2 , respectively, R denotes the gas constant, and ln signifies the natural logarithm.If the reaction is exothermic (ΔH° < 0), a rise in temperature will result in a decrease in the equilibrium constant, and conversely.This is because an increase in temperature causes the system to shift towards the side of the reaction with the lower heat content to offset the rise in heat energy.For an endothermic reaction (ΔH° > 0), the opposite holds true, with a temperature increase leading to an increase in the equilibrium constant.Essentially, at elevated temperatures, the equilibrium constant for the endothermic reaction C 6 H 12 O 6 + 2H 2 O → 2CH 3 COOH + 2CO 2 + 4H 2 will rise, indicating a higher concentration of products compared to reactants.This phenomenon occurs because the endothermic reaction absorbs heat as a reactant, and raising the temperature enhances heat absorption, thereby favoring the forward reaction and H 2 production.
Methanogenesis exhibits a greater tolerance for temperature fluctuations compared to the hydrogenogenic stage.This is particularly evident under psychrophilic (cold) and mesophilic (moderate) conditions (Gunes et al. 2019;Sasidhar et al. 2022).A slight temperature variation of ± 1 °C can adversely affect bioCH 4 production in thermophilic conditions, whereas mesophilic methanogens can withstand larger temperature fluctuations of up to ± 3 °C without significant decreases in bioCH 4 yields (Gunes et al. 2019).Thus, methanogenic reactors can be operated at ambient or mesophilic temperatures without requiring high-energy inputs for heating.

Retention time
In continuous-mode bioH 2 and bioCH 4 production, optimizing retention time within the anaerobic digester is critical.Hydraulic retention time (HRT) refers to the average duration the substrate remains in the digester, while solids retention time (SRT) denotes the average period bacteria are retained in anaerobic digesters (He et al. 2019).HRT is determined by the bioreactor volume, while SRT is influenced by the dominant microbial population in the bioreactor (Sasidhar et al. 2022).In traditional low-rate bioreactors without recycling or supernatant removal, SRT typically equals HRT.However, in high-rate bioreactors utilizing attached or suspended growth mechanisms, SRT is notably longer than HRT (Liu et al. 2011).
Methanogens and other bioH 2 consumers have a slow growth rate.Therefore, in hydrogenogenic reactors, shorter HRTs can help selectively enrich bioH 2 -producing bacteria and eliminate unwanted microorganisms (Roy and Das 2016).However, a drawback of shorter HRTs is the risk of cell washout, which can lead to decreased bioH 2 production (Wu et al. 2008).High-rate bioreactors like anaerobic fluidized bed reactors (AFBRs) have been successful in addressing this issue by achieving high biomass concentrations at very low HRTs (Ferreira et al. 2019).Extending the HRT may result in a shift in the bioH 2 formation pathway towards methanogenesis, potentially inhibiting bioH 2 production (David et al. 2019).In the methanogenesis stage, a longer HRT is generally recommended compared to the acidogenesis stage.This extended residence time, often around twice that required for slower-growing microbes, allows for several key benefits.First, it ensures sufficient time for the complete metabolization of complex organic compounds into simpler molecules.Second, it provides ample opportunity for the methanogenic microbial population to actively participate in the conversion process, maximizing bioconversion efficiency.Additionally, prolonging the HRT in methanogenic reactors can help eliminate dead zones and promote efficient syntrophy between microorganisms, especially in mixed cultures (Roy and Das 2016).
Overall, there is no set standard for determining the ideal HRT in two-stage bioH 2 and bioCH 4 production.It is dependent on several factors, including substrate properties, reactor configuration, process stage, and operating temperature (Kim et al. 2013).Literature shows a wide range of optimal HRT values for the first hydrogenogenic stage, from 3 h (Chu et al. 2022) to 10 days (Wirasembada et al. 2024), and for the second methanogenesis stage, from 3.5 h (Wu et al. 2016b) to 25 days (Dareioti et al. 2022;Sukphun et al. 2023a;Tsigkou et al. 2023).
SRT in two-stage AD processes has not been extensively studied.However, it is generally observed that the SRT for bioH 2 production is shorter (2-5 days) (Sasidhar et al. 2022) compared to bioCH 4 production (15-20 days) (Nabaterega et al. 2021).This difference is attributed to the quicker growth rates of hydrogen-producing microorganisms relative to methanogens.

Organic loading rate
A key factor for a stable and efficient AD process is the organic loading rate (OLR).It reflects the daily organic matter input relative to the digester's volume (Dangol et al. 2022).Increasing the substrate concentration can raise the OLR and enhance biogas production.However, excessive loading may lead to the accumulation of VFAs and acidification, potentially causing process failure (Grangeiro et al. 2019;Tyagi and Aboudi 2021).Conversely, if the reactor is underfed, it can become overly alkaline, reducing biogas productivity (Periyasamy et al. 2022).Therefore, it is crucial to conduct thorough assessments to determine the optimal OLR for the digester.
The optimal OLR for traditional single-stage AD systems typically falls within the range of 0.5 to 2 kg VS/m 3 /day, influenced by factors like feedstock characteristics, retention time, and operating temperature (Ramanathan et al. 2022).In contrast, two-stage AD systems, which separate the fermentation and methanogenesis phases, provide favorable conditions for distinct microbial communities.Research suggests that these systems can accommodate organic loads up to 300% higher than single-stage AD systems (Rajendran et al. 2020).Additionally, studies have shown that strategically distributing the OLR within a continuous two-stage system can improve biohythane production.This involves allocating a higher OLR to the first stage for bioH 2 production and a lower OLR to the second stage for bioCH 4 production (Montiel Corona and Razo-Flores 2018).OLR and HRT are interconnected parameters in anaerobic digestion, with the determination of one parameter dependent on the definition of the other.Consequently, adjusting one parameter can be achieved by fine-tuning the other as necessary (Sasidhar et al. 2022).

Nutrients
The success of the AD process relies on a balanced supply of macro and micronutrients.These vital nutrients support the complex biochemical activity of microbial communities and their associated enzymatic machinery, which are essential for the process.Nitrogen, phosphorus, and sulfur are essential macronutrients.Adequate nitrogen levels are necessary for microbial protein synthesis as it is a key component of amino acids (Choi et al. 2020).Nitrogen also helps maintain a stable reactive biosystem by providing sufficient buffering capacity (Krakat et al. 2017).However, high nitrogen concentrations in bioreactors can negatively affect process stability and efficiency due to the formation of ammonia, which can disrupt microbial function.Ammonia can enter microbial cells, causing proton imbalance and changes in intracellular pH.Additionally, free ammonia can inhibit specific enzymatic reactions (Morozova et al. 2020).Therefore, high ammonia concentrations can hinder both fermentative bioH 2 production and methanogenesis processes.While the reported optimal concentration of ammonia for bioH2 production may vary in different studies, a suggested range of 0.01-7 g/L based on available literature data (Bisaillon et al. 2006) can be considered ideal.Studies have shown that ammonia concentrations between 0.05 and 0.2 g/L can have a positive impact on the AD process (McCarty 1964).Critical ammonia concentrations that can lead to methanogenesis inhibition have been reported to range from 1.7 to 14 g/L (Chen et al. 2008b).Optimizing the carbon-tonitrogen (C/N) ratio within the digester is crucial for both minimizing ammonia concentration and maximizing biogas production.The range of 20 to 30 is widely considered the optimal C/N ratio for the AD process (Yen and Brune 2007;Zhang et al. 2014).Additionally, ensuring adequate phosphate levels is critical for maximizing biohythane production efficiency, as phosphorus is a vital mineral nutrient for microbial growth and function (Lei et al. 2010).Phosphate also plays a vital role in energy transfer and buffering capacity within the biohythane production system, acting as a substitute for carbonate (Chandrasekhar et al. 2015).However, high phosphorus levels, particularly at 3.3 mg/L, can negatively affect methanogenesis by increasing VFA production and lowering pH.Acidogenesis, on the other hand, is less impacted by these conditions (Mancipe-Jiménez et al. 2017).Sulfur is another crucial macronutrient for microorganisms involved in biohythane production, especially methanogens, commonly found in sulfate form in various feedstocks like food processing and pulp and paper waste (Dhar et al. 2012).Elevated sulfate concentrations can hinder dark fermentation and methanogenesis by promoting competition between bioH 2 and bioCH 4 producers and sulfate-reducing bacteria for organic matter, as well as causing toxicity from sulfide release (Chen et al. 2008b;Bundhoo and Mohee 2016).The optimal sulfur concentration for bioCH 4 production in anaerobic digestion is typically 1-25 mg/L (Chen et al. 2008b).Limited information is available on the impact of sulfate concentration on dark fermentation, but some studies suggest that 3000 mg/L sulfate at pH 5.5 may be effective for bioH 2 production (Lin and Chen 2006;Chen et al. 2008a;Hwang et al. 2009Hwang et al. , 2011)).High dissolved sulfide concentrations (> 100 mg/L) can inhibit bioH 2 production during dark fermentation, while concentrations of 100-800 mg/L are inhibitory to methanogens (Chen et al. 2008b;Dhar et al. 2012).
In addition to macronutrients, micronutrients like specific light and heavy metal ions are crucial for the AD process.
These micronutrients facilitate the synthesis and function of enzymes and coenzymes essential for bioH 2 and bioCH 4 production.These micronutrients also play a vital role in energy and electron transfer as well as microbial cell growth (Shanmugam et al. 2021b).While the right concentrations of light and heavy metals can promote microbial growth, it is crucial to be aware that excessive amounts can have the opposite effect, potentially leading to toxicity or inhibition of microorganisms (Chen et al. 2008b).Furthermore, each micronutrient contributes uniquely to microbial metabolism, and changes in its bioavailability can disrupt this process (Chandrasekhar et al. 2015).Therefore, it is crucial to regulate the levels of these micronutrients carefully to promote optimal microbial growth and avoid any adverse effects.Table 6 provides an overview of the key functions and recommended concentrations of important metal ions in both dark fermentative.

Potential feedstocks for biohythane production
The economic feasibility of the biohythane production process is closely tied to the use of renewable and cost-effective substrates that require minimal pre-treatment.While easily biodegradable simple sugars like glucose, sucrose, and starch have a high potential for biogas production, their expensive purchase price often makes this approach economically inefficient.To overcome this issue, replacing these materials with renewable organic feedstocks, especially biowaste, is widely acknowledged as a cost-efficient and sustainable strategy for biohythane production.By using biowaste as a feedstock, the process can benefit from lower substrate costs and the added bonus of waste management, making it an appealing choice for economic viability and environmental sustainability.

Organic fraction of municipal solid waste
Studies have extensively explored the organic fraction of municipal solid waste (OFMSW), particularly food waste, as a promising substrate for biohythane production (Sasidhar et al. 2022).The makeup and properties of these wastes differ based on their source and the season they are produced.In winter, food residues typically have higher protein content, while in summer, they are rich in carbohydrates due to reduced meat consumption and increased fruit and vegetable consumption (Bolzonella et al. 2018).Food waste also contains lipids, which produce higher-quality biogas compared to carbohydrates and proteins (Dasa et al. 2016).However, anaerobic digesters may face operational challenges due to lipid-induced clogging (Cirne et al. 2007).The breakdown of lipids leads to the formation of long-chain fatty acids (LCFAs), which can impede mass transfer by binding to microbial cell walls (Rinzema et al. 1994;Rasit et al. 2015).Additionally, fat adhesion can lead to biomass flotation and the loss of active biomass through washout (Hwu et al. 1998).
Fruit and vegetable wastes have a higher C/N ratio compared to meat residues, and their combination with other food waste portions influences the overall C/N ratio of the substrate (Bolzonella et al. 2018).High levels of lignin and cellulose in fruit and vegetable wastes can pose challenges for dark fermentation and methanogenesis, ultimately reducing biohythane yield (Meena et al. 2020).Managing pH levels is a key challenge when treating diverse substrates like food wastes (Kumar et al. 2021).
The range of biohythane production from various substrates in this category is wide, with values ranging from 23.6 mL/g VS for catering waste (Elwakeel et al. 2023) to 911.9 mL/g VS for OFMSW (Alavi-Borazjani et al. 2022).This variability highlights the significant impact of substrate type and operational conditions on biohythane yield.For instance, organic solid waste from a regional market yielded 258 mL/g VS of biohythane (Salazar-Batres et al. 2024), while household solid waste produced 543 mL/g VS (Liu et al. 2006).Catering waste, despite its low value of 23.6 mL/g VS in batch conditions, showed a biohythane production of 120.94 mL/g VS in continuous mode (Elwakeel et al. 2023).The range for OFMSW varies from 343 (Yeshanew et al. 2018) to 911.9 mL/g VS (Alavi-borazjani et al. 2022).Additionally, raw and processed food waste exhibit significant variability.Raw food waste values range from 212.88 (Cheng et al. 2016) to 669 mL/g VS (Chu et al. 2008).Processed food waste examples include food waste hydrolysate at 395.16 mL/g VS (Pomdaeng et al. 2024) and pre-aerated protein-rich food waste at 356.96 mL/g VS (Rafieenia et al. 2017).

Agricultural residues
Agricultural residues, including stalks, leaves, husks, and other organic materials from crops, serve as valuable and renewable substrates for biohythane generation.Despite their different physical appearances, all types of lignocellulosic biomass have a consistent chemical composition, including cellulose (30-70%), hemicellulose (15-30%), lignin (10-25%), and hydrophilic/lipophilic extractives (Ahmad et al. 2018).These substrates are rich in nitrogen and carbohydrates, making them suitable for maintaining the optimal C/N ratio required for biohythane production (Abdur Rawoof et al. 2021).However, the challenge lies in the rigid three-dimensional structure of these feedstocks, which hinders their biodegradation (Levin et al. 2009).Hence, it is crucial to employ efficient pre-treatment techniques, including physical, chemical, physicochemical, and biological processes, to break down the intricate composition of these feedstocks and enhance biogas production efficiency (Dar et al. 2021).
The biohythane yields from combining bioH 2 produced during the first stage and bioCH 4 generated in the second stage of AD processes treating agricultural residues exhibit considerable variation.For instance, tomato plant residues can yield 264 mL of biohythane per gram of VS (Ruiz-Aguilar et al. 2022).Wheat straw, a common agricultural residue, has shown a biohythane production of 396 mL/g VS (Kongjan et al. 2011).Sweet sorghum, as a substrate, has produced 39.4 mL of biohythane per gram of substrate (Antonopoulou et al. 2008).Date fruit wastes are particularly potent, generating 611 mL/g VS of biohythane (Saidi et al. 2023).Moreover, sugarcane straw hemicellulose hydrolyzate has been used to produce 288 mL/g COD of biohythane (Tomasini et al. 2023).These results highlight the significant potential of various agricultural residues in biohythane production, emphasizing their role as sustainable and efficient feedstocks in renewable energy generation.

Distillery/brewery wastes
Distilleries and breweries produce significant amounts of waste, posing environmental concerns because of the elevated levels of organic matter, nutrients (phosphorus, ammonia), and heavy metals (copper) (Dionisi et al. 2014).However, these waste streams can also be utilized as valuable resources for bioenergy production, especially for biohythane production, given their rich organic content (Roy and Das 2016).
Investigating the potential of distillery/brewery wastes for biohythane recovery, stillage, a common by-product in distilleries, has shown significant promise.Studies have shown that stillage can yield a combined bioH 2 and bioCH 4 yield of 417 mL/g VS (Luo et al. 2011).Furthermore, a mixture of stillage and excess sludge, tested under batch conditions, resulted in a biohythane production of 227 mL/g VS, while continuous conditions increased the biohythane production to 413 mL/g VS (Wang et al. 2011).Cassava stillage, derived from the fermentation and distillation processes involving cassava roots, has also shown potential in biohythane generation with a combined bioH 2 and bioCH 4 yield of 306 mL/g VS (Luo et al. 2010).Additionally, a combination of vinasse with spent brewer yeast resulted in a biohythane production of 258 mL/g VS (Nualsri et al. 2024).
Despite their rich organic content, distillery and brewery wastes have a complex and varied structure that makes them difficult for microbes to break down.Additionally, certain waste streams like pot ale contain high protein content, which can lead to increased ammonia concentrations within the bioreactor.These elevated ammonia levels have the potential to disrupt or even halt biogas production (Meena et al. 2020).Nevertheless, effective pre-treatment techniques and process adjustments can improve biohythane production from these plentiful waste sources, promoting the advancement of sustainable and eco-friendly bioenergy systems.

Dairy processing wastes
Dairy processing produces various liquid and solid by-products and wastes.Whey, the main by-product of the dairy industry, presents challenges for disposal because of its high because of its high nitrogen content and levels of biological and chemical oxygen demand (BOD and COD, respectively) (Kozłowski et al. 2019).However, whey is mainly made up of lactose (around 70%), a fermentable carbohydrate that can be turned into biogas (Meena et al. 2020).It also contains lipids, proteins, soluble vitamins, and minerals (Ahmad et al. 2019), making it a valuable resource for bioenergy production.
A recent trend in biofuel research focuses on harnessing these abundant waste materials to generate bioH 2 and bioCH 4 , the key components of biohythane.For example, Kovalev et al. (2022) demonstrated that two-stage AD of cheese whey yielded 36.86 mL bioH 2 /g COD in the initial stage, with an additional 325 mL bioCH 4 /g COD in the second stage, resulting in a total production of 361.86 mL/g COD.Similarly, Moreno-Andrade et al. ( 2015) found 1.11 mole bioH 2 /mole lactose during the initial stage and observed 170 mL bioCH 4 /g COD in the subsequent phase.Another two-stage approach achieved a bioH 2 yield of 0.78 mole /mole glucose consumed , followed by the generation of 147 mL of bioCH 4 /g VSS (Venetsaneas et al. 2009).Additionally, de Souza Almeida et al. ( 2023) conducted a study on biohythane production using a combination of cheese whey and glycerin.They found that in the first stage, bioH 2 production reached 9.9 mole/kg COD removed .In the second stage, bioCH 4 production was measured at 14 mole/ kg COD removed , resulting in a total biohythane yield of 23.9 mole/kg COD removed .
This in-depth analysis emphasizes the potential of utilizing dairy processing wastes, specifically cheese whey, for biohythane production.However, the use of low-alkaline substrates like cheese whey poses a challenge due to their tendency to undergo rapid acidification, leading to reduced biogas production or process inhibition (Meena et al. 2020).To address this issue, strategies such as incorporating costeffective buffering additives, integrating alkaline-rich cosubstrates, and implementing advanced monitoring systems can help mitigate the negative effects of rapid acidification and ensure the economic viability of bioenergy production from low-alkaline substrates such as cheese whey.

Livestock wastes
Livestock wastes generated in farms and slaughterhouses contain substantial amounts of protein, lipids, COD, volatile solids (VS), and low carbohydrate content (Sittijunda et al. 2010).The elevated COD level and microbial composition in these wastes make them highly polluting, and their direct disposal can lead to environmental issues (Meena et al. 2020).Recently, livestock waste has been acknowledged as a valuable raw material for producing bioH 2 and bioCH 4 because of its plentiful availability and rich organic matter content.However, as mentioned earlier, the significant lipid content in these wastes can cause biomass flotation and subsequent washout (Cirne et al. 2007).Moreover, lipid degradation leads to the formation of glycerol and long-chain fatty acids, which can accumulate and hinder microbial activity (Moukazis et al. 2018).Conversely, inhibitory effects from a high concentration of ammonia may arise due to protein degradation (Cuetos et al. 2008).
To address these challenges and improve biohythane production, these feedstocks are commonly co-digested with other substrates.This strategy not only helps to maintain nutrient balance but also improves the overall performance of the AD process.Several studies have demonstrated the potential of co-digestion in optimizing biohythane recovery from livestock wastes.For example, a combination of cow dung and untreated domestic wastewater sludge in a batch operation achieved a biohythane production of 877 mL/g VS.Similarly, mixing cattle slurry with grass silage produced 288 mL/g VS biohythane in a batch operation (Ning et al. 2023b) and 248 mL/g VS in a continuous feeding mode (Ning et al. 2023a).Furthermore, the mixture of poultry manure, wine vinasse, and sewage sludge in a continuous operation yielded a biohythane production of 431 mL/g VS by combining the bioH 2 and bioCH 4 values from the first and second stages of AD (Cheng et al. 2016).Additionally, codigestion of chicken manure, corn straw, and food waste led to a biohythane yield of 632.52 mL/g VS in a batch process (Liu et al. 2023).

Vegetable oil processing wastes
The vegetable oil extraction industry produces a substantial amount of by-products that hold potential for biohythane production.However, using this type of feedstock is not always feasible due to the sensitivity of anaerobes to fatrich substrates and intermediate compounds resulting from their decomposition (Hidalgo and Martín-marroquín 2014).These residues are typically acidic and contain macronutrients like nitrogen and phosphorus, as well as phenolic compounds that are resistant to biodegradation (Abdur Rawoof et al. 2021).These residues are rich in long-chain fatty acids, which can disrupt mass transfer processes at low concentrations due to their interaction with cell membranes (Hidalgo and Martín-Marroquín 2014).However, recent studies have reported microbial adaptations to long-chain fatty acids (Chen et al. 2008b).Despite these challenges, numerous studies have investigated the biohythane potential of various vegetable oil processing wastes.For example, olive pulp has shown promise in biohythane production, producing 0.13 mole bioH 2 / kg TS and 0.16 L bioCH 4 /kg COD (Koutrouli et al. 2009).Similarly, palm oil mill effluent has demonstrated significant biohythane production capabilities, with a yield of 535 L/kg COD (Krishnan et al. 2014).Additionally, the combination of palm oil mill effluent and concentrated latex wastewater has shown substantial biohythane production potential, with 95.45 mL bioH 2 /g VS and 1.20 mL bioCH 4 /g VS, leading to a total biohythane production of 96.65 mL/g VS (Raketh et al. 2023).These examples highlight the diverse potential of vegetable oil processing wastes in biohythane production, highlighting the versatility and effectiveness of these feedstocks.

Algal biomass
Algal biomass, found in both unicellular (microalgae) and multicellular (macroalgae) forms, is becoming increasingly popular for biohythane production.Algae have minimal lignin content, making them highly susceptible to microbial degradation (Montingelli et al. 2016).The energy potential of marine biomass, including algae, is estimated to be over 100 EJ per year, surpassing terrestrial biomass (22 EJ per year) and organic wastes (7 EJ per year) (Chynoweth et al. 2001).Furthermore, algae boast a remarkable potential for CO 2 , as the photosynthetic efficiency of aquatic biomass is reported to be 6-8%, approximately four times greater than that of terrestrial biomass (Aresta et al. 2005;Thompson et al. 2019).Additionally, algae cultivation boasts rapid growth rates and the potential for utilization on nonagricultural or marginal lands.They can even thrive using brackish water or wastewater, thereby reducing the need for competition with food crops for freshwater and fertilizers (Levine et al. 2010;Vivekanand et al. 2012).Notably, algal biomass for biogas production can be naturally harvested from eutrophic and degraded water bodies (Dȩbowski et al. 2013).
While the mentioned advantages make algal biomass an attractive option for biohythane production, there are also some drawbacks to consider.For example, specific algae strains with a high nitrogen content (low C/N ratio) may lead to the build-up of ammonia and VFAs in the digester, posing a challenge to efficiently harnessing this plentiful natural resource for biogas production (Zhong et al. 2012).However, this issue can be addressed by co-digesting highprotein algal biomass with a carbonaceous substrate to improve biogas production (Chen et al. 2015).Furthermore, biopolymers such as sporopollenin and algaenan in the rigid cell walls of algae can inhibit hydrolytic enzyme activity, thereby reducing biogas production rates (Kabir et al. 2022).Therefore, appropriate pre-treatment methods are essential to break down these biopolymers and enhance biohythane productivity.

Other feedstocks
In addition to the previously discussed categorized feedstocks, various other substrates have been investigated for their potential in biohythane production.The biohythane production values for these feedstocks vary widely and are influenced by different factors.For instance, sedimented pulp and paper mill waste fiber have shown promise, with a study reporting a combined bioH 2 and bioCH 4 production of 376.1 mL/g VS (El-Qelish et al. 2024).Similarly, tofu-processing residue, also known as okara, has exhibited significant biohythane production capabilities.One study reported a biohythane production of 114.4 mL/g VS, comprising 50 mL bioH 2 /g VS and 64.4 mL bioCH 4 /g VS (Ali et al. 2024), while another study demonstrated a higher biohythane yield of 581.42 mL/g VS (Ali et al. 2022).
Residual glycerin, a by-product of biodiesel production, has also shown promise with a biohythane yield of 976 mL/ L medium (de Oliveira Faber et al. 2023).Moreover, corn steep liquor, a by-product of corn wet milling, has demonstrated potential in biohythane recovery with a bioH 2 production of 670 cm 3 /L/day and a bioCH 4 production of 220 cm 3 /L/ day (Stoyancheva et al. 2023).Halophytic biomass, such as Atriplex crassifolia, has also been researched, yielding 13.2 mL bioH 2 /g in the initial stage and 8.5 mL bioCH 4 /mL in the subsequent second stage (Nawaz et al. 2023).Napier grass has been another focus, showing a biohythane production of 457 mL/g VS (Pomdaeng et al. 2022).
Furthermore, skim latex serum desulfated by rubber wood ash has demonstrated potential, with a biohythane yield of 367.56 mL/g COD, comprising 73.03 mL bioH 2 /g COD and 294.53 mL bioCH 4 /g COD (Raketh et al. 2022).Additionally, research on cassava starch-based polymers has revealed a biohythane production of 269 mL/g VS (Cremonez et al. 2020).Water hyacinth leaves, a type of invasive plant, have shown 195.1 mL/g VS of biohythane, including 51.7 mL bioH 2 /g VS and 143.4 mL bioCH 4 /g VS (Cheng et al. 2010).Lastly, investigations into spent mushroom beds have resulted in a biohythane production of 252.49mL/g VS of biohythane (Bertasini et al. 2024).Table 7 provides a comprehensive overview of previous studies on two-stage bioH 2 and bioCH 4 (biohythane) production from various substrates.

Bioreactor design/configurations
Recent research provides strong evidence in favor of utilizing a sequential two-stage AD process instead of a single-stage approach for maximizing biohythane production (Sasidhar et al. 2022).Therefore, to achieve the highest bioH 2 and bioCH 4 yields in the two-stage process and to enable largescale industrial biohythane production, the effective integration of bioreactors is crucial.
The selection of bioreactor configuration in the two-stage biohythane production process is influenced by various factors, especially the properties of the feedstock utilized (Rajendran et al. 2020).Figure 6 depicts the potential reactor integration strategies proposed for two-stage biohythane production, which are dependent on the feedstock concentration.For feedstocks with a high total solid (TS) content exceeding 15% (dry basis), a Leaching Bed Reactor (LBR) is a suitable choice for the initial biohydrogen production stage.The resulting leachate can then be processed in highperformance anaerobic reactors such as the Up-flow Anaerobic Sludge Blanket (UASB), Up-flow anaerobic packedbed (UAPB), and expanded granular sludge bed (EGSB) for bioCH 4 generation in the subsequent stage (Liu et al. 2013).When the feedstock's TS falls within the moderate range of 2-12%, continuous stirred-tank reactors (CSTRs) are generally considered the preferred option for bioH 2 production.For the next methanogenic stage in this case, different reactor configurations can be chosen depending on the characteristics of the digestate produced in the initial stage.Regarding substrates with a very low TS content (less than 2%), high-rate anaerobic reactors like UASB can be employed for both biohydrogenation and biomethanation stages (Abdur Rawoof et al. 2021).
In practice, different combinations of reactors have been used in two-stage AD systems.While batch mode may be easier to operate and more economically feasible, continuous mode has been shown to be more efficient (Meena et al. 2020).Among bioreactor types, CSTR stands out as the most commonly employed for both bioH 2 and bioCH 4 production stages (Mozhiarasi et al. 2023) due to its advantages such as ease of setup and capacity to facilitate direct contact between the substrate and active biomass through complete mixing (Nageswara-Rao and Soneji 2018).For example, Cavinato et al. (2011) demonstrated the effectiveness of a two-stage CSTR system operating at thermophilic conditions (55 °C) using municipal biowaste as a substrate.The first stage, prioritizing bioH 2 production, employed a shorter HRT (3.3 days) and a higher OLR (16 kg VS/m 3 /d), achieving a bioH 2 yield of 51 L/kg VS.In contrast, the second stage focused on maximizing bioCH 4 yield by using a longer HRT (12.6 days) and a lower OLR (4 kg VS/m 3 /d), resulting in a significant increase in bioCH 4 production to 416 L/kg VS.Similarly, potato waste was processed using a two-stage system with CSTRs at mesophilic conditions (35 °C).The initial stage, with an initial pH of 5.5 and an HRT of 6 h, yielded 30 L bioH 2 /kg TS, while the second stage controlled at a neutral pH of 7 and an HRT of 90 days produced 183 L bioCH 4 /kg TS (Zhu et al. 2008).For a combination of wine vinasse, sewage sludge, and poultry manure, an integrated two-stage system using CSTRs showed that operating the first stage at 55 °C with an HRT of 5 days achieved 40.41 mL bioH 2 /g VS.The second stage operated at 35 °C with an HRT of 12 days, yielded 391 mL bioCH 4 /g VS.Additionally, a combination of CSTR and UASB reactor was employed in some studies.For instance, in a UASB-CSTR system treating palm oil mill effluent, the first UASB stage operated at 55 °C and an HRT of 2 days, yielding 215 L bioH 2 /kg COD, while the subsequent CSTR stage at 37 °C with an HRT of 5 days generated 320 L bioCH 4 /kg COD (Krishnan et al. 2014).A two-stage approach utilizing a CSTR followed by a UASB reactor was also explored for Agave tequilana bagasse (Montiel Corona and Razo-Flores 2018).In the initial CSTR stage maintained at 35 °C, a shorter HRT of 6 h and a higher OLR of 44 g COD/L/d facilitated bioH 2 production, resulting in a bioH 2 yield of about 6 L/L/d.Subsequently, the process transitioned to a UASB reactor operating at a lower temperature range (23-25 °C) with a longer HRT of 14 h and a reduced OLR of 20 g COD/L/d, achieving a bioCH 4 yield of approximately 6.4 L/L/d.
Apart from the aforementioned systems, numerous other reactor configurations have proven effective in two-stage AD processes.For example, Salazar-Batres et al. ( 2024) investigated a two-stage SBR-SBR (Sequencing Batch Reactor) system for treating organic solid waste.Both stages were maintained at a constant temperature of 37 °C.The first stage operated with a 16-h HRT and a high OLR of 60 g VS/L/d, promoting bioH 2 production at 28 mL/g VS.The subsequent stage utilized a longer HRT of 2.8 days and a lower OLR of 2.8 g VS/L/d, focusing on bioCH 4 generation with a yield of 230 mL/g VS.Additionally, synthetic wastewater was processed using an SBBR-SBBR (Sequencing Batch Biofilm Reactor) configuration.The first SBBR operated at 28 °C           2023) investigated a two-stage AFBR (Anaerobic Fluidized Bed Reactor) system using a cheese whey-glycerin mixture.The initial phase was conducted at 55 °C with a 4-h HRT and 11.6 kg COD/m 3 /d OLR, resulting in 9.9 moles of bioH 2 / kg COD removed .The second phase, operating at 30 °C with a 24-h HRT and an 18.5 kg COD/m 3 /d OLR, produced 14 moles of bioCH 4 /kg COD removed .Table 8 outlines the main advantages and disadvantages of bioreactors utilized in previous studies for biohythane production.Substrates with a resistant structure significantly reduce the hydrolysis rate, hindering biohythane production.Therefore, pre-treatment of these substrates is crucial to enhance their biodegradability.Researchers have proposed various approaches for this purpose, including physical, chemical, and biological methods.However, choosing the appropriate pre-treatment method for these challenging substrates is still a difficult task.This is because each method has its own strengths and weaknesses, making it difficult to find a universal approach that can effectively treat all types of feedstocks.
Accurate monitoring and control of pH are essential for efficient bioH 2 production and subsequent bioCH 4 generation.In laboratory settings, commercial chemicals are commonly used for pH control.While adding external chemicals for pH control may be straightforward in smallscale biohythane production setups, it becomes more difficult and costly in large-scale plants.Excessive alkali addition in such operations can result in a digestate with a high salt concentration, reducing its potential as a biofertilizer.Additionally, using chemicals for pH regulation can lead to high concentrations of metal ions and ammonia, presenting further challenges (Krishnan et al. 2019).Therefore, it is crucial to explore alternative approaches for pH control in large-scale operations to address these issues and ensure efficient and sustainable biohythane production.
It is essential to purify biogas from both the hydrogenogenic and methanogenic stages to ensure that only pure bioH 2 and bioCH 4 gases are suitable for use in the biohythane blend.Several techniques have been developed for biogas purification over the years, with some currently in use on an industrial scale.However, the economic viability of biohythane production is hindered by the high energy and chemical requirements of existing purification methods.Therefore, it is essential to prioritize the development of sustainable and energy-efficient approaches for biohythane purification to address these challenges and establish a more economically feasible pathway for biohythane production.
In addition to the points mentioned above, there are significant challenges related to the use of biohythane and the handling of by-products from the AD process.A key obstacle is the current gas distribution system and the lack of developed filling stations, which hinder the widespread commercialization of biohythane.The absence of enforced standards further complicates the situation, highlighting the need for regulatory frameworks and guidelines to facilitate its market integration (Mozhiarasi et al. 2023).Managing digestate, the primary by-product of biohythane production, is also a major challenge.While there is potential to reuse digestate as a soil fertilizer or nutrient source for the hydrogenation stage, various obstacles must be addressed, including land constraints and potential negative effects on

Main challenges for biohythane scale-up
While there are numerous benefits to biohythane production using the AD process, it is important to acknowledge and address the various limitations associated with this technology.Overcoming these challenges is crucial for the successful implementation of biohythane production on a larger scale, allowing for the efficient recovery of biohythane from the substantial volumes of organic waste produced globally.Figure 7 illustrates the main challenges in scaling up the biohythane production process, with further explanations provided below.
BioH 2 is an intermediate product of biomethanation, and to recover it as part of biohythane, the hydrogenotrophic methanogenesis pathway must be inhibited (Liu et al. 2018;Krishnan et al. 2019).A key challenge in achieving this lies in managing the microbial community, particularly during the initial hydrogenogenic phase (Mozhiarasi et al. 2023).However, using mixed microflora in the fermentation process may be inefficient due to bioH 2 consumers like hydrogenotrophic methanogens, requiring pre-treatment measures and making the process economically unfeasible (Shanmugam et al. 2021a).Selecting an appropriate pre-treatment method poses technical challenges as not all bioH 2 producers can form endospores. Conversely, some bioH 2 consumers, like acetogens, can form spores, adding complexity to the process (Krishnan et al. 2019).While using pure cultures has been effective in enhancing bioH 2 production, it is not practical for largescale implementation (Krishnan et al. 2019).Therefore, using mixed microbial consortia is preferred over pure cultures to reduce costs.However, ensuring the availability of mixed consortia rich in bioH 2 -producing microorganisms during scale-up is essential.
The efficient integration of the hydrogenogenic and methanogenic stages is crucial for successful biohythane production.The functioning of the bioCH 4 -producing reactor relies on the bioH 2 -producing reactor, where the used medium from the initial phase acts as the methanogens' substrate in the subsequent phase.Nonetheless, transferring feedstock continuously between the first-and second-stage reactors poses a challenge, requiring a sophisticated control system (Mozhiarasi et al. 2023).Moreover, additional energy inputs are required for tasks like heating, mixing, and pumping, and energy losses can occur due to phase changes and product release (Liu et al. 2018).Overall, the separate production of bioH 2 and bioCH 4 in distinct bioreactors is technically complex and involves high investment, operating, and maintenance costs (Abdur Rawoof et al. 2021).Therefore, integrating the two stages into a single digestion unit can help alleviate these challenges significantly (Shanmugam et al. 2021a).the bioH 2 -producing microbial consortium from long-term recirculation (Krishnan et al. 2019).To address these challenges, a comprehensive approach is required.This involves developing a reliable gas distribution network with wellequipped filling stations for efficient biohythane delivery.Additionally, establishing enforceable standards and certifications is essential to ensure the safety, quality, and compatibility of biohythane with existing infrastructure.Regarding digestate management, exploring innovative strategies, such as advanced treatment processes and targeted land application techniques, can maximize its value as a resource.Implementing advanced monitoring and control strategies for the recirculation process can help mitigate potential negative impacts on the bioH 2 -producing microbial consortium, ensuring the efficiency and stability of the AD system over the long term.

Techno-economic and environmental assessments
Conducting a techno-economic assessment is a critical step in the commercialization of biohythane production via the AD process, as it offers valuable insights into the project's profitability and feasibility (Hans and Kumar 2019).The total project cost comprises the total capital investment and the total annual cost (operating cost) (Jarunglumlert et al. 2018).The total capital investment (continued) includes the fixed capital costs of constructing the plant, which encompasses equipment, installation, engineering, construction, and working capital expenses incurred in setting up and operating the plant until revenue is generated (Vo et al. 2018).On the other hand, the total annual cost is the sum of all expenses accrued during the production process, such as raw materials, labor, maintenance, laboratory costs, and so forth (Jarunglumlert et al. 2018).
The annual revenue of the biohythane production plant is derived from the sale of biogas and solid waste, as well as waste treatment fees (Jarunglumlert et al. 2018).Once the total costs and annual revenue are estimated, the project's profitability performance indicators can be determined (Hans and Kumar 2019).
While there is ample research on cost estimates for hydrogen production using various methods such as steam methane reforming, coal and biomass gasification, and electrolysis (Khan et al. 2018), information on the costs of producing bioH 2 through dark fermentation is limited and requires further investigation.Nevertheless, the production, refining, and bottling of bioH 2 have similarities with bioCH 4 (Sasidhar et al. 2022), which could offer insights into cost projections.Therefore, the scalability of biohythane production largely hinges on capital costs, which can potentially be reduced through government incentives like energy efficiency schemes (Mozhiarasi et al. 2023).By providing subsidies and tax benefits, the government can alleviate the financial burden on investors and incentivize them to invest in biohythane production.The initial investment required to establish a two-stage AD process for treating 13.4 tons per hour of biowaste was estimated at 12,687.7 k€ (Ljunggren and Zacchi 2010).Furthermore, research conducted by Micolucci et al. (2018) revealed that processing 27 tons of biowaste per day with a dual-stage thermophilic AD system could yield an annual income of 540,874 €, equivalent to approximately 54.9 € per ton.The payback period for these projects generally ranges from 2 to 6 years, influenced by factors such as the specific composition and properties of the feedstock used in the process (Mozhiarasi et al. 2023).
Another crucial factor that can significantly influence the economic viability of biohythane production is the production scale (Han et al. 2016).As production scale increases, the capital investment cost per unit of production decreases due to economies of scale.This decrease in capital cost can result in a lower overall production cost and a higher profit margin.However, larger-scale production necessitates a greater supply of feedstock, which can raise transportation costs and potentially influence the quality and consistency of the feedstock.Therefore, determining the optimal production scale that balances the advantages of economies of scale with the costs associated with feedstock supply and transportation is essential for the commercial success of biohythane production.
The decision to adopt a two-stage AD process for biohythane production involves balancing the energy output with the added cost of a second digester.Research has shown that the two-stage process yields a positive energy balance compared to single-stage processes for bioH 2 or bioCH 4 production, even when factoring in pre-treatment and reactor heating energy requirements (Mozhiarasi et al. 2023).Further, the additional capital investment required for the dual-stage AD in comparison to the single-stage approach is reported to be only 3% (Rajendran et al. 2020), which can be well compensated by the extra energy produced.
In addition to evaluating the economic feasibility of biohythane production, analyzing potential environmental impacts is crucial for its large-scale commercialization.Limited environmental assessments have been conducted on biohythane production via the AD process.In a study by Lembo et al. (2022), the carbon footprints of three different anaerobic digester configurations treating second cheese whey were compared.These configurations included a conventional single-stage anaerobic digester located 50 km from the dairy factory, an on-site single-stage anaerobic digester within the dairy industry premises, and an on-site two-stage system for bioH 2 and bioCH 4 recovery.The research findings indicated that the two-stage AD process demonstrated superior energy output, resulting in a 60% reduction in greenhouse gas emissions compared to the off-site single-stage AD.This reduction was primarily attributed to increased bioH 2 production and enhanced engine performance.Patterson et al. (2013) used an LCA approach to assess the environmental burden associated with the two-stage bioH 2 and bioCH 4 production from wheat feedstock, compared to single-stage bioCH 4 production.The results indicated that the two-stage process had a reduced environmental footprint.They also found environmental advantages in using a bioH 2 -bioCH 4 blend from the two-stage process as a vehicle fuel over diesel.Sinsuw et al. (2024) conducted a detailed LCA on two-stage biohythane production from livestock wastes at commercial and pilot scales, considering various impact categories.They found that photochemical ozone creation potential had the highest total impact at the commercial scale (33%), while eutrophication potential had the highest impact at the pilot scale (33%).Both biogas production systems had low overall environmental impacts, with the commercial system showing lower impacts than the pilot system.The use of digesters significantly reduced potential environmental impacts related to manure feedstock handling and fertilizer applications.Chen et al. (2020) also utilized an LCA approach to comprehensively assess the energy conversion characteristics and environmental impacts of two-stage biohythane production from microalgae and food waste.The total greenhouse gas emissions from the system were quantified at 173 g CO 2-eq MJ −1 .The main contributors to these emissions were electricity production (41.6%),CO 2 release during pressurized water processes (27.8%), and energy recovery (19.8%).The carbon fixation process by microalgae significantly decreased net greenhouse gas emissions to 124 g CO 2-eq MJ −1 .Additionally, variations in microalgae growth rate and biohythane yield were identified as key factors influencing greenhouse gas emissions.
Previous studies have also shown that the environmental impacts and advantages of biohythane vary depending on how it is used.For example, research by Liu et al. (2018) employing LCA methodology discovered that biohythane derived from the AD process of corn stalks, when utilized as a vehicle fuel, exhibits a more favorable impact on greenhouse gas emissions compared to alternative options such as combined heat and power (CHP), direct combustion, and compression.Moreover, Bolzonella et al. (2018) discovered that the solid-liquid digestate from the dual-stage biohythane production system has fewer environmental concerns such as acidification and eutrophication due to its lower levels of acid, nitrogen, and phosphorus compared with the conventional single-stage bioCH 4 production approach.
The techno-economic and environmental assessments discussed above highlight the significant promise of biohythane production through the AD process for sustainable energy and waste management solutions.It is crucial to balance capital investments with operational costs to achieve profitability, with potential reductions possible through government incentives.Moreover, scaling up biohythane production shows clear benefits in terms of economies of scale, despite challenges in feedstock logistics and quality control.Environmental assessments reveal substantial reductions in greenhouse gas emissions and other environmental impacts with the adoption of two-stage AD systems, demonstrating their superiority over conventional methods.These findings underscore the critical role of thorough assessments in guiding the development and deployment of biohythane technologies toward broader commercial viability and environmental sustainability.

Biohythane integration in waste management systems
The practical application of biohythane production through anaerobic digestion shows great potential for improving waste management practices and providing economic and environmental advantages.However, integrating this technology into existing waste management systems requires careful consideration of several key factors:

Infrastructure and technological integration
The existing waste management facilities primarily rely on landfill disposal or traditional biogas production, highlighting the necessity for substantial improvements in infrastructure and technology.This involves the implementation of effective pre-treatment units for sorting and treating organic waste, utilizing advanced anaerobic digesters that can produce both bioH 2 and bioCH 4 , and employing sophisticated gas separation and purification systems to maintain the desired biohythane composition.Addressing these technical requirements is crucial for seamlessly incorporating the new systems into the current infrastructure.

Economic viability
Assessing the economic feasibility of biohythane production requires a thorough techno-economic analysis.This assessment includes considering initial capital investments, ongoing operational expenses, and market factors affecting hydrogen and methane.Furthermore, potential income from selling hydrogen and methane, as well as government incentives for renewable energy generation and waste disposal, can improve economic feasibility.

Regulatory and policy framework
Effective regulatory frameworks are essential for the successful implementation of renewable energy solutions.Governments can encourage the production of renewable energy, set quality standards for market acceptance, and implement policies that promote waste diversion to biohythane facilities, thereby reducing the dependence on landfills.

Environmental impact
Biohythane production supports sustainability objectives by decreasing greenhouse gas emissions and encouraging resource recovery.By redirecting organic waste from landfills to a controlled anaerobic digestion process, methane emissions can be greatly reduced.Biohythane production also allows for the recovery of valuable resources from waste, promoting a circular economy.Furthermore, the concurrent generation of bioH 2 and bioH 2 increases the overall energy output from organic waste, offering a more efficient option compared to conventional biogas production.

Community engagement and awareness
Active community involvement is vital for the successful adaptation of biohythane technology.Public acceptance and participation can be fostered through education and outreach programs that inform communities about the benefits of biohythane and encourage their involvement in waste segregation and collection efforts.Collaboration with local governments, industries, and non-profit organizations can build a supportive network for advancing biohythane production.
By addressing these multifaceted aspects, biohythane is poised to have a significant impact on the waste management sector, providing a sustainable route to a more environmentally friendly future.Moving forward requires conducting pilot projects to demonstrate feasibility, investing in research and development to optimize processes, advocating for supportive policies, and providing capacity building for professionals to effectively manage biohythane production systems.This comprehensive strategy ensures a holistic approach to realizing the full potential of biohythane production.

Future prospects and possible improvements
The future of biohythane production is poised for significant advancements in efficiency, sustainability, and scalability.Key areas for improvement include:

Enhanced pre-treatment methods
Effective pre-treatment methods are crucial for converting diverse biomass feedstocks into biohythane.Enzymatic pretreatment, while environmentally friendly, needs further optimization to address cost and time constraints.Microwave and ultrasound pre-treatment methods show promise for their efficiency, but scalability remains a challenge (Aashabharathi et al. 2024).Future research could focus on developing innovative pre-treatment approaches tailored to different feedstock compositions, optimizing energy efficiency, and reducing processing costs.

AD process optimization
Optimizing biohythane production involves refining both single-stage and two-stage AD processes.This includes understanding metabolic pathways, scaling-up technical processes, and enhancing reactor performance.Continuous refinement and comprehensive evaluations are essential for commercial-scale biohythane production.Innovations in bioreactor design, especially in integrating advanced monitoring and control systems, can significantly enhance production efficiency.Artificial intelligence (AI) and machine learning (ML) are powerful tools in this endeavor, designed solely on historical data and real-time process information without the need for mathematical models or human intervention.They hold promise, especially in intricate industrial processes where creating mathematical models and obtaining human expertise is challenging (Kazemi et al. 2021).AI-based algorithms like artificial neural networks (ANN) and genetic programming (GP) have proven successful in optimizing process parameters, predicting product yields, and addressing AD issues (Almomani 2020;Gonçalves Neto et al. 2021;Andrade Cruz et al. 2022).Potential future developments could involve the integration of AI and ML for real-time optimization and the development of modular bioreactor systems for decentralized biohythane production.

Microbial community regulation
Unlocking the full potential of biohythane production requires in-depth knowledge and manipulation of the microbial consortia involved in the process.However, there is a significant lack of comprehensive meta-omics research on AD microbial communities.Multi-omic approaches, such as metagenomics, metatranscriptomics, metaproteomics, and metabolomics, show promise in elucidating microbial functionalities and dynamics, but they necessitate advanced analytical methods and specialized software (Sukphun et al. 2023b).Furthermore, gene manipulation and bio-augmentation strategies are crucial for optimizing AD processes.Cutting-edge genome editing tools like CRISPR/Cas9 and CRISPR/AsCas12a allow for precise genetic modifications (Li et al. 2022;Hao et al. 2023), opening up new possibilities for enhancing AD efficiency and increasing biohydrogen and methane production.Additionally, bio-augmentation, as demonstrated by the development of microbial consortia such as KKU-MC1, has shown potential in enriching microbial communities and enhancing biogas output from challenging biomass sources (Wongfaed et al. 2023).Nevertheless, further research is needed to evaluate the feasibility of bio-augmentation for commercial applications and its integration into the full-scale biohythane production process.

Utilization of digestate by-products
Leveraging the potential of digestate by-products presents opportunities for resource recovery and circular economy practices.Approaches like composting, nutrient extraction, and biochar production can convert digestate into valuable resources, reducing waste and promoting sustainability (Hung et al. 2017;Vaneeckhaute et al. 2017;Ezemagu et al. 2021).Future efforts could focus on creating integrated biorefinery models to optimize resource extraction from digestate, leading to reduced environmental impact and fostering the sustainable utilization of biomass resources.

Gas purification, storage, and distribution
Efficient gas purification techniques are essential for maintaining the quality and safety of biohythane.Future advancements could concentrate on creating decentralized purification systems, investigating renewable energy-driven purification technologies, and improving gas storage methods.The utilization of hybrid purification systems, like the membrane-amine hybrid system, can enhance purification effectiveness, decrease energy consumption, and reduce operational expenses.These hybrid systems combine the advantages of various separation processes, addressing the drawbacks of each and enabling more efficient removal of contaminants from the gas stream (Abdulsalam et al. 2019).Furthermore, exploring the on-site integration of hydrogen and methane as a potential method to optimize biohythane quality and usability could be beneficial in the future.This strategy would provide greater control over the gas composition, potentially enhancing the performance and versatility of biohythane in various energy applications.It is also crucial to expand distribution networks to reach remote or underserved areas.Future initiatives should concentrate on expanding infrastructure, integrating smart grid technologies for improved distribution management, and ensuring the reliability and efficiency of biohythane supply chains.

Techno-economic and lifecycle analyses
While existing evidence suggests the economic viability and environmental benefits of biohythane production, further upscaling studies and comprehensive environmental assessments are necessary.Future research should prioritize refining cost projections and exploring environmental advantages across diverse feedstocks and applications to bolster the advancement and adoption of biohythane production technologies.Recent progress has seen the successful integration of advanced modeling techniques in both techno-economic analysis (Sampat et al. 2022) and lifecycle assessment (Khalaj et al. 2023), enhancing the accuracy and reliability of these evaluations.Future investigations could utilize these sophisticated modeling approaches to analyze the dynamic interactions among technological advancements, market conditions, and policy frameworks.This approach will facilitate informed decision-making and risk management for biohythane projects, ensuring their economic feasibility and environmental sustainability.

Policy support and market expansion
Supportive policies and incentives are necessary to foster the growth of biohythane as a viable energy source.Measures like investment subsidies, promoting biohythane energy projects, and developing strong supply chains can help expand the market and encourage consumer acceptance.Future policy initiatives could concentrate on creating clear regulatory structures, encouraging research and development in biohythane technologies, and promoting international cooperation to standardize practices and facilitate global trade.These actions would contribute to creating a supportive environment for sustained market growth and competitiveness.

Conclusion
Biohythane is a promising next-generation biofuel, created by combining bioH 2 and bioCH 4 from single-stage or twostage AD processes.Various microorganisms play key roles in the production of biohythane, each with specific growth and activity requirements.Optimization of parameters such as temperature, pH, retention time, organic loading rate, and nutrient content is crucial for maximizing biohythane yield.Effective recovery is also influenced by substrate type and bioreactor configuration.While the two-stage AD process offers superior energy recovery compared to single-stage methods, challenges like high costs and maintenance impede its widespread commercialization for biohythane production.To achieve success, addressing technical hurdles such as managing microbial consortia, integrating bioH 2 and bioCH 4 generation processes, monitoring pH levels, and selecting appropriate pre-treatment methods for complex substrates is essential.Cost-effective biogas purification methods and addressing constraints related to biohythane and by-product utilization are also critical.Furthermore, conducting comprehensive techno-economic and environmental assessments is essential to evaluate the feasibility and sustainability of biohythane production processes.These assessments are pivotal for informing strategic decisions and advancing the viability of biohythane as a renewable energy source.

Fig. 5
Fig. 5 Inoculum pre-treatment methods for enrichment of hydrogenproducing bacteria (optimum: 98 °C) -Ability to thrive in highly saline environmentsKurr et al. (1991);Forterre (2006);Garcia et al. (2006); Angelidaki et al. (2011); Oren (2014c) Methanocella Methanocellales -Rod-shaped, coccoid -Hydrogenotroph -Mesophilic to thermophilic conditions (optimum: 35-55 °C) -Activity preference at extremely low H substrate-to-inoculum ratio, IRPR internal recirculation packed-bed reactor, LBR leaching-bed reactor, SBR sequencing batch reactor, IABR integrated anaerobic bioreactor, PABR periodic anaerobic baffled reactor, AnSTBR anaerobic structured-bed reactor, TDAPR thermophilic down-flow anaerobic packed-bed reactor, SCRD semi-continuous rotating drum, AFBR anaerobic fluidized bed reactor, AnSBBR anaerobic sequencing batch biofilm reactor, ZVI zero-valent iron with a higher OLR of 4.75 kg COD/m 3 /d compared to the second bioreactor, resulting in the production of 1.59 moles of bioH ₂ /kg COD removed .The second stage, also operating at 28 °C but with an OLR of 1.81 kg COD/m 3 /d, yielded 0.363 m 3 of bioCH 4 /kg COD removed (Venkata Mohan et al. 2008).In another study, synthetic wastewater was treated using a two-stage mesophilic system with suspended-growth reactors in both stages.In the first stage, HRT and SRT were both set to 1 day, with a feed rate of 0.225 L/d.This stage resulted in a bioH 2 production of 5.64 mL/g COD.The second stage, with an HRT and SRT of 20 days and a feed rate of 0.2 L/d, produced 320 mL of bioCH 4 /g COD (DiStefano and Palomar 2010).Furthermore, wheat straw underwent treatment in a dual-stage UASB configuration.The initial phase functioned at 70 °C with a 1-day HRT and 9.9 g VS/L/d OLR, achieving a bioH 2 production of 89 mL/g VS.The second stage UASB functioned at 55 °C with a 3-day HRT and 3.3 g VS/L/d OLR, resulting in a bioCH 4 yield of 307 mL/g VS(Kongjan et al. 2011).Additionally, de Souza Almeida et al. (

Fig. 6
Fig. 6 Proposed reactor integrations for sequential bioH 2 and bioCH 4 generation based on the feedstock concentration (adapted and redrawn from Liu et al. (2013)).ABR anaerobic baffled reactor, CSTR continuously stirred tank reactor, EGSB expanded granular sludge bed reactor, LBR leaching-bed reactor, UAPB up-flow anaerobic packed-bed reactor, UASB upflow anaerobic sludge blanket reactor setup and operation -Uniform mixing -Enhancing mass transfer and direct contact between substrate and active biomass -Effective for washing out methanogens in the first hydrogenogenic stage -Long retention time and large reactor volume -Energy intensive -Potential disruption of microbial flocs at high rotational speeds -Risk of biomass washout at short HRTs -Risk of short-circuit currents and flushing out undigested substrate without proper stirring Skiadas et al. (2003); Show et al. (2011); Nageswara-Rao and Soneji (2018); Hans and Kumar (2019); Alavi-Borazjani et al. (2020); Martinez-Burgos et al. (2021); Dangol et al. (2022); Holl et al. (2022); Singh et al. (2023) UASB -Equipment versatility -Low operational cost -Suitable for feedstocks with low TS content -Retaining high biomass concentration -Handling high OLRs -Low sludge production -Long start-up time -No guarantee of success for the self-immobilization process -Vulnerability to sudden shock loads -Filtration requirement for large particles when treating certain substrates -Requirement for effluent post-treatment Veronez al. (2005); Tiwari et al. (2006); Show et al. (2011); Abdur Rawoof et al. (2021); Martinez-Burgos et al. (2021); Holl et al. (2022) LBR -Suitable for feedstocks with high TS content -Low energy and water demand -Effective in separating dissolved organic compounds -Preventing the loss of partially decomposed substrate and active biomass -Leachate recycling without utilizing the solid-liquid separation unit -Inefficient hydrolysis of complex feedstocks -Sophisticated operational condition control -Diminished leachate permeability or bed clogging at elevated OLRs Liu et al. (2013); Dangol et al. (2022); Holl et al. (2022); Singh et al. (2023) ASBR -Simple design -No need to external filtration -Providing long retention times for solids -No short circuit -Biomass retention without using fixed media or a solid settling chamber -No need to primary and secondary settles -Possibility of intermittent operation -Efficient operating control -Highly stable under hydraulic shock loads -Pressure variations during feeding and withdrawal -Precise agitation requirements -Challenging to decant -Risk of reactor structure damage due to negative pressure Sarti et al. (2007); Pinheiro et al. (2008); Shao et al. (2008); Yang et al. (2019); Holl et al. (2022)

Fig. 7
Fig. 7 Main challenges for scaling up the biohythane production process

Table 1
Global renewable energy capacity in 2023 and percentage increase from 2014

Table 3
Advantages and disadvantages of single-stage and two-stage AD

Table 4
Microorganisms involved in biohydrogen production

Table 6
Main functions and optimal concentrations of essential metals in bioH

Table 8
Advantages and disadvantages of various bioreactors employed in biohythane production